Connexin-hemichannels are Involved in Acidosis-induced ATP Release from Skeletal Myocytes

Abstract

ATP is an important extracellular signalling molecule which contributes to exercise vasodilation. We have previously shown that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in acidosis-induced ATP release from skeletal muscle. However, it is still unknown whether ATP is released through CFTR itself or whether CFTR regulates a separate ATPrelease channel. So we investigated: (1) the pathway responsible for CFTR activation in myocytes at low pH; (2) whether connexin (Cx) hemichannels were involved in the acidosis-induced ATP release from skeletal muscle. Lactic acid (10 mM) increased the intracellular cAMP and the extracellular ATP in L6 skeletal myocytes. Similarly, the cAMP-elevating agent, forskolin, increased extracellular ATP. The phosphodiesterase inhibitor, IBMX, increased extracellular ATP in the absence or presence of lactic acid. CFTR phosphorylation was increased by the addition of forskolin alone, and further increased by forskolin plus dibutyryl-cAMP and IBMX, but the forskolininduced increase in CFTR phosphorylation was inhibited by the PKA inhibitor, KT5720. Whereas KT5720 inhibited acidosis-induced ATP release from myocytes. These data suggest that skeletal muscle CFTR is activated through the cAMP/PKA pathway at low pH. RT-PCR indicated that cultured rat L6 skeletal myocytes expressed mRNA for both Cx40 and Cx43, but Cx40 was expressed only weakly in western blot, whereas Cx43 was strongly expressed. Co-immunoprecipitation results showed that CFTR and Cx43 were associated with each other in the cell membrane. A Cx43 over-expression model was created by transfecting myocytes with a Cx43 plasmid: Cx43 over-expression was confirmed using western blot. Cx43 over-expressing myocytes released significantly more ATP than control myocytes at pH 6.8, suggesting that Cx43 may be involved in acidosis-induced ATP release, whereas silencing Cx43 expression using siRNA inhibited the acidosis-induced ATP release. Over-expression of CFTR alone did not alter ATP release from myocytes, whereas co-over-expression of CFTR with Cx43 increased ATP release significantly more than over-expression of Cx43 alone. These data suggest that Cx43 co-localises with CFTR in the myocyte membrane, and that it may be involved in ATP release during acidosis; further investigation is required to determine whether and how CFTR interacts with Cx43 to induce ATP release.